PROJECT SUMMARY During the last funding period, Project 1, headed by Dr. Fabio Cominelli, in close collaboration and synergy with Project 2 and 4, has made important discoveries regarding the role of NOD2/CARD15 in the pathogenesis of experimental Crohn's disease (CD). This competitive renewal will continue our efforts to investigate intestinal innate immunity in the pathogenesis of experimental ileitis. The central hypothesis of Project 1 is that the interaction(s) between NOD receptor signaling, the gut microbiome, and IL-1 play a key role in the pathogenesis of chronic intestinal inflammation. To test this hypothesis we will: 1) Determine the role of NOD1 and RIP2 signaling in experimental CD. Using a series of cross-breeding experiments with our SAMP and TNF?ARE mice, we will define the role of NOD1 and RIP2 in early versus late phases of chronic ileitis. By using bone marrow chimera experiments, we will determine the contribution of hematopoietic and non-hematopoietic- derived NOD1 and RIP2 signaling in mediating CD-like ileitis. Finally, congenic and bone marrow chimera SAMP mice will be challenged with DSS administration to induce chronic CD-like colitis and will be compared to control mice for severity of chronic intestinal inflammation and cytokine production. 2) Determine the role of the gut microbiome in mediating NOD2 signaling activation in experimental CD. We will first characterize the composition and function of the microbiome in SAMPxNOD2-/- mice compared to SAMPxNOD2+/+ controls by 16S RNA and meta-transcriptome analysis. In addition, using a series of fecal material transfer (FMT) experiments of gut microbiome from genetically-manipulated SAMP mice, as well as patients with NOD2 mutations, we will precisely define the role of NOD-induced dysbiosis in the pathogenesis of chronic ileitis. 3) Determine the role of IL-1 family members (IL-1?, IL-1?, and IL-1Ra) in the pathogenesis of CD-like ileitis. We have excellent preliminary results showing that specific neutralization of IL-1? markedly ameliorates the severity of CD-like ileitis in SAMP mice with potency equivalent to steroids. We will first determine the mechanism(s) regulating IL-1?, IL-1? and IL-Ra in tissue and mucosal cells isolated from mice with CD-like ileitis. We will then perform a series of antibody neutralization studies specifically blocking IL-1?, IL-1?, and a combination of the two in SAMP and TNF?ARE mice. We will then investigate how IL-1? and IL- 1? regulate the number and function of ILCs in both SAMP and TNF?ARE CD-like ileitis (in collaboration with Project 3). Through these specific aims, Project 1 will systematically evaluate the role of NOD receptors, the gut microbiome, and IL-1 in mediating the CD-like pathology found in SAMP and TNF?ARE mice. The overall objective of Project 1 is to develop new therapeutic modalities through manipulation of intestinal innate immune responses that either prevent or generate a ?permanent? remission in patients affected by this devastating disease.